Alcohol is the most commonly used recreational and abused substance. While beneficial effects of moderate alcohol use on coronary heart disease exists, prolonged excessive alcohol use predisposes to infection and results in liver and other organ diseases. Studies from our and other laboratories demonstrated that acute alcohol use attenuates immune responses while prolonged alcohol exposure augments inflammatory responses. The overall goal of this research proposal is to define molecular mechanisms that regulate the switch from the acute, anti-inflammatory to the chronic, pro-inflammatory effects of alcohol. We propose that the opposite effects of acute and prolonged alcohol treatment are related to differences in activation of the Toll-like receptor (TLR)4-induced MyD88-dependent and MyD88-independent signaling pathways. We hypothesize that inhibition of monocyte inflammatory responses to LPS stimulation after acute alcohol exposure is a result of alcohol-induced LPS tolerance. Specifically, we propose that acute alcohol induces tolerance to LPS stimulation via its affects on MyD88-dependent TLR4 signaling. We postulate that in contrast to acute alcohol, prolonged alcohol exposure results not only in loss of TLR4 tolerance but also in sensitization of LPS-induced activation of pro-inflammatory pathways in monocytes/macrophages. We hypothesize that sensitization to LPS involves activation of the MyD88-independent pathway that will amplify MyD88-independent signals in cells that lost TLR tolerance after prolonged alcohol exposure. As an extension of these hypotheses, we propose that prolonged alcohol exposure results in sensitization to monocyte activation though TLRs other than TLR4 which relates to host interaction with a broad variety of pathogens. The aims of this proposal are: 1. To evaluate whether the opposite effects of acute and chronic alcohol on LPS-induced monocyte/macrophage activation are related to TLR4 tolerance after acute and/or to loss of TLR4 tolerance after prolonged alcohol exposure by investigating negative regulators and molecular signatures of TLR tolerance. 2. To evaluate the role of TLR4-induced, MyD88-independent signaling in sensitization to LPS-induced inflammatory cytokine activation by prolonged alcohol. 3. To determine the effects of acute and chronic alcohol treatment on monocyte pro-inflammatory activation by selective MyD88-utilizing TLRs (other than TLR4) involved in recognition of bacterial (TLR2 and TLR5), and viral (TLR7/8) pathogen-derived danger signals Results from these experiments will delineate important molecular markers of inflammatory cell inhibition by acute and activation by chronic alcohol which may provide targets for future therapeutic interventions. PUBLIC HEALTH RELEVANCE: Alcohol is the most commonly used recreational and abused substance. While beneficial effects of moderate alcohol use on coronary heart disease exists, prolonged excessive alcohol use predisposes to infection and results in liver and other organ diseases. We propose to study monocytes, that are important cells in inflammation, to test the hypothesis that acute and prolonged alcohol use have opposite effects on inflammation through specific effects on cell activation through the pathogen recognition receptor, Toll-like receptor 4. Investigation of both acute and chronic alcohol responses in human monocytes represents a unique and clinically applicable approach to better understand human conditions modified by alcohol intake including coronary heart disease and alcoholic liver diseases.